Idiopathic pulmonary fibrosis (IPF) is an inexorably progressive disease, but the rate of progression is variable and at times can be catastrophic. The mechanisms that regulate abrupt acceleration or rapid progression of disease are completely unknown. We have reported that aberrant innate sensing of hypomethylated DNA in lung myofibroblasts drives the rapid progression of fibrosis via endosomal TLR9 activation. More recently, we have discovered that a cytoplasmic and nuclear DNA sensor known as DNA- protein kinase (DNA-PK) is also present in human primary fibroblasts, and this sensor is also involved in the responsiveness of IPF fibroblasts to a hypomethylated DNA (i.e. CpG-DNA) signal. Additional preliminary data we have generated suggest that innate DNA sensors promote myofibroblast activation and accelerate fibrosis via a pathway that involves type 1 interferon alpha (IFN?), growth arrest specific-6 (Gas6), and Axl (a Gas6 receptor). Using this framework of published and unpublished data, the present grant will address the following specific hypothesis: hypomethylated DNA drives the activation of DNA sensors in primary human pulmonary fibroblasts leading to myofibroblast differentiation and activation via a targetable IFN?- Gas6-Axl receptor-dependent mechanism. Finally, we have data suggesting that this process is a vicious cycle in which Gas6/Axl interactions drive the expression of TLR9 and DNA-PK in IPF fibroblasts. Thus our proposed studies will focus on the following Specific Aims: 1) Determine the role of innate DNA sensors in the activation of normal and IPF fibroblasts. 2) Determine the role of type 1 IFN? in the activation of normal and IPF fibroblasts. 3) Determine the role of Gas6-Axl in the activation of normal and IPF fibroblasts. We will examine primary human fibroblasts propagated from either stable and progressive forms of IPF or normal lung samples for the presence of DNA sensors and their downstream signaling components. The primary human fibroblasts will be interrogated for the signaling mechanism(s) via which innate DNA sensing drives myofibroblast differentiation, synthetic activity, and invasiveness (i.e. migratory behavior). Finally, we will use a humanized model of IPF initiated by the intravenous introduction of primary human fibroblasts into immunodeficient mice with the goal of further interrogating and therapeutically targeting the DNA sensing mechanisms leading to rapid fibrosis in vivo. Thus, this translational proposal will address the molecular mechanisms through which primary human fibroblasts are activated by exogenous hypomethylated DNA, and will seek to identify therapeutic strategies that interrupt this process during the exacerbation or rapid progression of pulmonary fibrosis.